Compared to other phospholipids, PIP2 is a highly negatively charged lipid. At physiological pH, it has a valence of−4. Apart from PIP3, other phospholipids only have a valence of−1.
Additionally, different from other lipids, the valence of PIP2 depends on many factors, such as local pH, proteins binding to it and local ion concentration (McLaughlin et al., 2002). In Fig. 1.3A the structure of PIP2 is illustrated. It has been suggested that due to its charge and structure, PIP2 penetrates further into the aqueous phase than other phospholipids (McLaughlin et al., 2002). The concentration of PIP2 within a cell was calculated as 10µM, which is equivalent to 1% of phospholipids (Gamper and Shapiro, 2007b).
1.5.1 Functions of PIP2
PIP2 regulates many different cellular processes: exo- and endocytosis, membrane traffick-ing, protein trafficktraffick-ing, phagocytosis, activation of enzymes, receptors and channels (Fig.
1.3, McLaughlin et al., 2002; McLaughlin and Murray, 2005). It serves as a second messen-ger precursor, giving rise to IP3, DAG and also PIP3. These molecules are present in a low concentration in quiescent cells and can increase in concentration upon receptor activation.
They are therefore ideal as second messengers. For example, the binding of phospholipase Cδ1 (PLCδ1) to PIP2 localizes it to the plasma membrane. Upon receptor activation, PLC is activated and hydrolyses PIP2 to IP3 and DAG, which leads to a rise in intracellular Ca2+. Increased intracellular Ca2+concentration results in activation of various enzymes. Thereby, PLCδ1 is not activated through its binding to PIP2 but through binding to Ca2+. The mem-brane anchorage through the binding to PIP2 simply facilitates hydrolysis. Furthermore, a direct link between PIP2 and proteins that bind to cytoskeleton has been shown to influence the membrane tension and therefore the shape of cells (Raucher et al., 2000). Additionally, PIP2 was shown to be involved in exocytosis and clathrin-mediated endocytosis. Various studies have indicated that PIP2 is involved in the insertion and uptake of membrane and is therefore important in regulating plasma membrane morphology (Mellman, 2000; Golub and Caroni, 2005). Membrane trafficking can therefore influence the cell shape and the composition of the plasma membrane. Taken together, PIP2 is thought to organize mem-brane extension and overall cell shape. PIP2 was also shown to bind scaffolding proteins, and is involved in the regulation of ion channels (Hilgemann et al., 2001). Additionally, during phagocytosis, PIP2 is concentrated in nascent phagosome and membrane ruffles and was shown to play a role in initial cup formation (Yeung et al., 2006a). Recent findings have shown that PIP2 and PIP3 play a crucial role in apical membrane formation during epithelial cyst formation and axon specification (Martin-Belmonte et al., 2007). PIP3 and its precursor PIP2 might also play a major role in the polarization of oligodendrocytes. Dur-ing development, oligodendrocytes form processes that are later retracted. Oligodendrocyte polarization is therefore comparable to neuronal polarization (Simons and Trotter, 2007).
In neurons several initial processes are formed, before one of them receives a positive signal to extend, thereby sending a retractive signal to other processes. During the formation of
A
B
Figure 1.3: (A) Molecular structure of PIP2. PIP2 is formed through phosphorylation of 4’ and 5’ OH groups within the inositol ring mainly at the plasma membrane through PIPK type I activity. Thereby the overall charge of this phospholipid is reduced to −4 at a physiological pH. PIP2 is anchored to the membrane through two poly-unsaturated hydrocarbon chains.
(B) PIP2 is involved in almost all cellular processes. It regulates membrane extension and cell shape (through regulation of exo- and endocytosis, phagocytosis, membrane ruffles, cell motility, cell adhesion and it is involved in the capture of microtubules). It also plays a role in signal transduction pathways and serves as a second messenger precursor (IP3 and PIP3) as well as an activator of ion channels or receptors (Images modified from Di Paolo and De Camilli, 2006).
drives specification of the axon (Shi et al., 2003; Banker, 2003; Martin-Belmonte et al., 2007;
Goldstein and Macara, 2007). It was shown that PIP3 plays a crucial role in myelin for-mation in the central nervous system (Flores et al., 2008). Loss of the PIP3-phosphatase PTEN in oligodendrocytes of mutant mice leads to hypermyelination, indicating that an increase of PIP3 drives myelin formation. An increase of PIP3 in oligodendrocytes results in increased myelination, even in adult mice (Goebbels et al., unpublished observation).
1.5.2 Enzymes generating PIP2
Phosphorylation of different OH groups within the inositol ring through different PIPkinases leads to the generation of distinct phosphoinositols. The enzymes are located at distinct subcellular localizations (Anderson et al., 1999). Phospholipids are therefore inhomoge-nously distributed within the membrane system of the cells (Krauss and Haucke, 2007).
PIP2 is mainly generated upon phosphorylation of PI4P at the 5’ OH-group through the PIPK typeI and can be found at the plasma membrane. The PIP2 kinases are activated upon receptor stimulation and can lead to localized production of PIP2 (Doughman et al., 2003). The small GTPase Arf6 is also involved in PIP2 production, by activating PI4P5K, and induces the accumulation of PIP2 at the plasma membrane (Donaldson, 2003). The main phosphatases of PIP2 are Synaptojanin 1 and SHIP1. Synaptojanin 1 is involved in the regulation of PIP2 levels and is indispensable for PIP2 recycling during vesicle trafficking (Milosevic et al., 2005). Synaptojanin 1 knockout mice therefore display a developmental phenotype and fail to develop, mainly due to failure in endocytosis (Cremona et al., 1999).
PIP2 is thought to be more abundant in myelin compared to other membranes of other cell types. Studies of 32P incorporation into myelin have shown, that within 60 min of incu-bation about 15% of 32P-labeled PIP2 is incorporated into myelin (Deshmukh et al., 1981;
Kahn and Morell, 1988). Additionally it is believed that PO3− groups are provided by the axon. It is therefore not surprising that all PIP2 generating enzymes can be found in the myelin sheath (Chakraborty et al., 1999).
1.5.3 Protein domains binding to PIP2
There are several known protein domains that can bind PIP2: unstructured-domains, tubby, pleckstrin homology (PH); phox homology (PX); epsin N-terminal homology (ENTH) do-mains, four-point-one, ezrin, radixin, moesin (FERM) and many other. PH-domains bind with high affinity to PIP2, the PH-domain of PLCδ1 for example binds with Kd= 2µM.
It comprises 120 amino acids and binds to PIP2 in a 1:1 ratio (McLaughlin et al., 2002).
The binding of the unstructured domain (also called natively unfolded proteins) of ’myris-toylated alanine-rich C kinase substrate’ (MARCKS) to PIP2 has been under intensive investigation (reviewed in Sheetz et al., 2006). MARCKS binds several PIP2 molecules at the same time mainly through electrostatic interaction of distinct Lys residues. MARCKS was also shown to bind to Ca2+/CAM, PKC and actin. Phosphorylation through PKC and binding of Ca2+/CAM was shown to influence the binding of MARCKS to the plasma membrane. Several other unstructured domains were reported to bind to PIP2. Among them are actin-binding proteins, such as GMC (GAP43, MARCKS, CAP23), also referred to as PIPmodulins due to their ability to cluster PIP2 at the cell membrane (Laux et al., 2000).
1.5.4 Molecular tools to monitor phospholipids
Since recent years it has become apparent that phosphoinositides take part in the control of almost every aspect of a cell’s life. Recent findings have led to the demand of new technologies to visualize PIP2, in order to study the spatio-temporal aspects of inositide signaling. Since PH domains of various proteins bind with different affinity to distinct phosphoinositides, these lipids can be visualized by adding a fluorescent tag to these PH domains. While the PH domain of AKT has widely been used to visualize PIP3, the PH domain of PLCδ1 has been used to visualize PIP2. Additionally, FRET pairs of different PH domains have been used to study the activity of PLC (van der Wal et al., 2001). Upon PLC activation, PIP2 is hydrolyzed to IP3 and DAG, which results in the dissociation of GFP-PH-PLCδ1. Cotransfection of the FRET pair composed of PH-PLCδ1 with a CFP and YFP tag respectively therefore showed a decreased FRET efficiency upon PLC activation. These fluorescently tagged domains are therefore widely used to study dynamics and interactions
of phosphoinositides (van der Wal et al., 2001; Várnai and Balla, 2007). In this study I used different sensors to monitor the distribution and dynamics of phosphoinositides.
2 Materials and Methods
2.1 Materials
Chemical reagent were purchased from SIGMA, unless noted otherwise.
2.1.1 Cell Culture
2.1.1.1 Mammalian cell lines
COS-1 Green monkey kidney, fibroblast cells
Oli-neu Rat, O2A cells OLN-93 Rat, O2A cells
BHK Baby hamster kidney cells
2.1.1.2 Mammalian cell culture media
DMEM for mammalian cell culture was purchased from GIBCO or BioWhittaker, BME and OptiMEM from GIBCO.
BHK cell medium 2.5% Horse serum OptiMEM
10% Tryptose phosphate broth Hepes
Penicillin/Streptomycin (BioWhittaker) OLN-93 cell medium
DMEM 10% FCS
Penicillin/streptomycin
Oli-neu cell medium (SATO) Concentration Component
10µg/ml Insulin 1 µg/ml Transferrin 25µg/ml Gentamycin
220 nM Sodium-Selenite 520 nM L-Thyroxine 500 pM Tri-iodo-threonine 100 µM Putrescine 200 nM Progesterone DMEM 4,5 g/l glucose
Sterile filter and add between 1 to 5 % Horse Serum
Freezing Medium for all cell lines 70% DMEM
20% FCS (PAN Biotech) 10% DMSO
Primary oligodendrocyte medium A.before shake
BME 10% HS
Penicillin/Streptomycin B. after shake
Sato medium 1% HS
2.1.2 Strains and cells
2.1.2.1 Bacterial strains Escheria Coli
DH 5α XL1-Blue
2.1.2.2 Bacterial culture media
Prior to the use, bacterial media were autoclaved and supplemented with antibiotics.
LB-Medium
1 % Bacto Tryptone 0.5 % Bacto Yeast extract 1 % NaCl
Make 1000 ml with H2O, set pH 7.5 with 10 N NaOH and autoclave.
Antibiotics were used at the following concentrations:
150 mg/l Ampicillin 25 mg/l Kanamycin 50 mg/l Chloramphenicol
2.1.3 Molecular cloning reagents
2.1.3.1 Plasmids
pEYFP-N1 BD-Biosciences Clontech pECFP-N1 BD-Biosciences Clontech
pGEMT Promega
pEYFP-Mem Clontech
pMSCVhyg BD-Biosciences Clontech
pET22b(+) Novagen
pSFVgen provided by M. Simons
2.1.3.2 Enzymes
Pfu Stratagene
Dpn1 NewEngland Biolabs
Taq SIGMA
Easy-A DNA Stratagene
T4 ligase Promega
2.1.3.3 Buffers
DNA-sample buffer (6x)
20%(w/v) Glycerol in TAE buffer 0.025% (w/v) Orange G bromphenol blue
dNTP stock solution (100 nM)
25 mM each dATP, dCTP, dGTP, dTTP(Boehringer, Mannheim) 1µg/ml Ethidiumbromide for agarose gels in TAE
TAE (50x, 1000ml)
2 M Tris-Acetate, pH 8.0
50 mM EDTA
57.1 ml Glacial acetic acid make 1000 ml with ddH2O
2.1.3.4 Primer sequences and PCR protocol
Primer Sequence
exon 1 sense CCG GAA TTC GCC ACC ATG GCA TCA CAG AAG AGA
exon 1 antisense CGC GGA TCC TTG CCA GAG CCC CGC TT
Exon 1 MBP14k-YFPS54A sense CCC AAG CGG GGC GCT GGC AAG GAT C
Exon 1 MBP14k-YFPS54A antisense GGG TTC GCC CCG CGA CCG TTC CTA G
MBP14k-YFP full length S54A CCC AAG CGG GGC GCT GGC AAG GAC TCA CAC ACG AG
MBP14k-YFP full length S54A CTC GTG TTG TGA GTC CTT GCC AGC GCC CCG CTT GGG
PCR 4. 68◦C 6 min (amplification, 30 cycles to step 2) 5. 68◦C 10 min
SDS running buffer (1x) 25 mM TrisHCl 192 mM Glycin 1% (w/v) SDS SDS separating gel
12.0 % (1 gel of 1.5mm thickness)
4 ml 30% polyacrylamid (BioRad) 10 ml separation gel buffer
(1.5 M Tris-HCl; 0.4% (w/v) SDS), pH 8.8 3.5 m l ddH2O
SDS stacking gel
12% (1 gel of 1.5mm thickness)
0.8 ml 30% poly-acrylamid (BioRAD) 1.5 ml stacking gel buffer (0.5M Tris-HCl 0.4%(w/v) SDS, pH 6.8
3.7 ml ddH2O
20µl Ammonium persulfate (10% w/v)
20µl TEMED
2.1.4.2 Membrane isolation buffers TE (1x)
10 mM Tris-HCl, pH 8.0
1 mM EDTA
Sucrose gradient buffer 250 mM saccarose 3 mM imidazol
(add protease inhibitor tablets fresh) TNE
10 mM Tris-HCl, pH 7.4
0.2 M NaCl
1 mM EDTA
2.1.5 Immunofluorescence labeling reagents
mRFP-LactC2 provided by S. Grinstein, Toronto, Canada GFP-PH-PLCδ1
obtained from I. Milosevic and J. Soerensen (MPI for Biophysical Chemistry, Göttingen, Germany) GFP-PH-PLCδ1-3xmut
IPPCAAX-GFP-SFV(mRFP-Synj1)
PIP4P5K-GFP-SFV
ARF6/Q67L-HA provided by J.G. Donaldson, NIH, Bethesda, Maryland
PH-AKT-YFP provided by T. Meyer (Stanford, CA)
MBP cDNA provided by T. Campagnoni (UCLA, CA)
∆Exon1(MBP)-YFPÊ
provided by Angelika Kippert, University of Göttingen; department of Biochemistry II MBP14k-YFP
MBP21k-YFP
∆Exon(1,3,5)-YFP Exon7-YFP
2.1.7 Antibodies
NG2 mouse provided by J Trotter 1:50
MBP rabbit DAKO 1:200
MBP mouse DAKO 1:200
Tuj1 mouse Covance 1:500
Lamp1 mouse PharMingen 1:100
KDEL (BIP) mouse Stressgen Biotechnologies 1:100
GM130 mouse BD Transduction Laboratories 1:100
HA rat own production 1:50
Cy5 mouse Chemicon 1:250
cy3 rabbit Dianova 1:4000
cy3 mouse Dianova 1:2000
cy2 mouse Dianova 1:2000
O1(anti-GalC) mouse provided by J Trotter 1:50
O4(anti-sulfatide) mouse provided by J Trotter 1:50
Par3 rabbit provided by T Pawson 1:100
phospho-AKT rabbit Cell Signalling 1:200
2.1.8 Chemical compounds
Ionomycin - dissolved in DMSO, Calbiochem Wortmannin - dissolved in DMSO, Sigma
DMSO - Sigma
21.4 g Glyconerin ether, 1,2,3- propanetriol glycindyl ether